Sickle cell trait individuals usually have about 60% HbA and 40% HbS. Those who also have an alpha-thalassemia gene(s), which decreases production of alpha chains, have an even lower proportion (25-35%) of HbS. To provide a model for the effect of the alpha-thalassemia gene(s) on HbA and HbS levels, we measured the amounts of the two hemoglobins that were reconstituted from various mixtures of native alpha, beta A, and beta S chains. Heme-containing 3H-labeled alpha chains, 14C-labeled beta S chains, and unlabeled beta A chains were isolated by p-mercuribenzoate dissociation of corresponding hemoglobins, column chromatographic separations of the subunits, and mercaptoethanol treatment to remove completely the mercurial agent. Various amounts of 3H-alpha chains were added to replicate mixtures containing equal amounts of beta A and 14C-beta S chains. After incubation at 4 degrees for 60 minutes, the relative amounts of 3H-radioactivity as HbS and HbA were determined by electrophoresis on cellulose acetate strips. The amount of HbS formed was less than that of HbA when alpha chains were present in limiting amounts. At very low relative alpha chain levels (microgram alpha/microgram total beta less than 0.05) the ratio of HbS/HbA formed was constant at about 0.6. Conversely, when microgram alpha/microgram total beta greater than 1, HbS/HbA formed equals 1. The quantitative recovery of 14C-radioactivity as either HbS or residual free beta S chains showed that selective instability or loss of beta S chains was not responsible for the results. We suggest that there was a difference in amounts of HbS and HbA formed because of a difference in affinities of beta S and beta A chain monomers for alpha chains. Additional studies are in progress to determine if the enhanced deficit of HbS in sickle trait individuals with an alpha-thalassemia gene(s) occurs because of a similar mechanism.